Enzymes and structural proteins play important roles in human physiology as well as hold a potential usefulness in industrial and clinical applications, such as bioremediation and therapeutic drug design. Understanding how protein structure relates to function allows us to determine the role of abnormal proteins in disease, and is necessary for the success of protein engineering. The main goal of this proposal is to use a new combinatorial form of random mutagenesis or "directed evolution" called DNA shuffling to study how mutations in all regions of protein structure affect the specificity of ligand binding and catalysis. Rather than starting with preconceived hypotheses of what structural features of a protein affect these characteristics, this method allows nature to provide us with the solutions. Isocitrate dehydrogenase (IDH) will be randomly mutagenized and mutants that exhibit altered substrate specificity and can use isopropylmalate as a substrate will be selected using a bacterial growth assay. Because IDH is well characterized structurally and mechanistically, detailed analyses of the mutants by kinetic studies and crystallography will be possible. This will provide valuable insights into the structural basis of substrate specificity, and aid in future studies.